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The yeast Cyberlindnera jadinii is a close relative of Candida utilis that is being used in the food and feed industries. Here, we present the 12.7Mb genome sequence of C. jadinii strain CBS 1600 generated by next generation sequencing. The deduced draft genome sequence consists of seven large scaffolds analogous to the seven largest chromosomes of C. utilis. An automated annotation of the C jadinii genome identified 6147 protein-coding sequences. The level of ploidy for both genomes was analyzed by calling single nucleotide polymorphisms (SNPs) and was verified measuring nuclear DNA contents by florescence activated cell sorting (FACS). Both analyses determined the level of ploidy to diploid for C. jadinii and to triploid for C utilis. However, SNP calling for C. jadinii also identified scaffold regions that seem to be haploid, triploid or tetraploid. (C) 2015 Elsevier B.V. All rights reserved.

Pseudomonas aeruginosa is an opportunistic pathogen that typically infects patients with a compromised immune defense. Here, we present the improved 6.5-Mb draft genome of strain WS136, an ExoS-positive and ExoU-negative highly cytotoxic chronic wound isolate recovered from pyoderma gangrenosum of a patient who received bone marrow transplantation.

Pseudomonas aeruginosa is a frequent human pathogen that increasingly causes chronic infections of nonhealing wounds. Here we present the 6.8 Mb draft genome of strain WS394, a multidrug-resistant chronic ulcer isolate that exhibited outstanding high cell cytotoxicity despite defective secretion of exotoxin U, suggesting a habitat-dependent adaptation process.

With the widespread availability of high-throughput sequencing technologies, sequencing projects have become pervasive in the molecular life sciences. The huge bulk of data generated daily must be analyzed further by biologists with skills in bioinformatics and by “embedded bioinformaticians,” i.e., bioinformaticians integrated in wet lab research groups. Thus, students interested in molecular life sciences must be trained in the main steps of genomics: sequencing, assembly, annotation and analysis. To reach that goal, a practical course has been set up for master students at the University of Lausanne: the “Sequence a genome” class. At the beginning of the academic year, a few bacterial species whose genome is unknown are provided to the students, who sequence and assemble the genome(s) and perform manual annotation. Here, we report the progress of the first class from September 2010 to June 2011 and the results obtained by seven master students who specifically assembled and annotated the genome of Estrella lausannensis, an obligate intracellular bacterium related to Chlamydia. The draft genome of Estrella is composed of 29 scaffolds encompassing 2,819,825 bp that encode for 2233 putative proteins. Estrella also possesses a 9136 bp plasmid that encodes for 14 genes, among which we found an integrase and a toxin/antitoxin module. Like all other members of the Chlamydiales order, Estrella possesses a highly conserved type III secretion system, considered as a key virulence factor. The annotation of the Estrella genome also allowed the characterization of the metabolic abilities of this strictly intracellular bacterium. Altogether, the students provided the scientific community with the Estrella genome sequence and a preliminary understanding of the biology of this recently-discovered bacterial genus, while learning to use cutting-edge technologies for sequencing and to perform bioinformatics analyses.

Adduct formation, fragmentation events and matrix effects impose special challenges to the identification and quantitation of metabolites in LC-ESI-MS datasets. An important step in compound identification is the deconvolution of mass signals. During this processing step, peaks representing adducts, fragments, and isotopologues of the same analyte are allocated to a distinct group, in order to separate peaks from coeluting compounds. From these peak groups, neutral masses and pseudo spectra are derived and used for metabolite identification via mass decomposition and database matching. Quantitation of metabolites is hampered by matrix effects and nonlinear responses in LC-ESI-MS measurements. A common approach to correct for these effects is the addition of a U-13C-labeled internal standard and the calculation of mass isotopomer ratios for each metabolite. Here we present a new web-platform for the analysis of LC-ESI-MS experiments. ALLocator covers the workflow from raw data processing to metabolite identification and mass isotopomer ratio analysis. The integrated processing pipeline for spectra deconvolution “ALLocatorSD” generates pseudo spectra and automatically identifies peaks emerging from the U-13C-labeled internal standard. Information from the latter improves mass decomposition and annotation of neutral losses. ALLocator provides an interactive and dynamic interface to explore and enhance the results in depth. Pseudo spectra of identified metabolites can be stored in user- and method-specific reference lists that can be applied on succeeding datasets. The potential of the software is exemplified in an experiment, in which abundance fold-changes of metabolites of the l-arginine biosynthesis in C. glutamicum type strain ATCC 13032 and l-arginine producing strain ATCC 21831 are compared. Furthermore, the capability for detection and annotation of uncommon large neutral losses is shown by the identification of (γ-)glutamyl dipeptides in the same strains. ALLocator is available online at: https://allocator.cebitec.uni-bielefeld.​de. A login is required, but freely available.

The growing amount of information resulting from the increasing number of publicly available genomes and experimental results thereof necessitates the development of comprehensive systems for data processing and analysis. In this paper, we describe the current state and latest developments of our BRIGEP bioinformatics software system consisting of three web-based applications: GenDB, EMMA and ProDB. These applications facilitate the processing and analysis of bacterial genome, transcriptome and proteome data and are actively used by numerous international groups. We are currently in the process of extensively interconnecting these applications. BRIGEP was developed in the Bioinformatics Resource Facility of the Center for Biotechnology at Bielefeld University and is freely available. A demo project with sample data and access to all three tools is available at https://www.cebitec.uni-bielefeld.de/groups/brf/software/brigep/. Code bundles for these and other tools developed in our group are accessible on our FTP server at ftp.cebitec.uni-bielefeld.de/pub/software/.

Ten different antibiotic resistance plasmids conferring high-level erythromycin resistance were isolated from an activated sludge bacterial community of a wastewater treatment plant by applying a transformation-based approach. One of these plasmids, designated pRSB101, mediates resistance to tetracycline, erythromycin, roxythromycin, sulfonamides, cephalosporins, spectinomycin, streptomycin, trimethoprim, nalidixic acid and low concentrations of norfloxacin. Plasmid pRSB101 was completely sequenced and annotated. Its size is 47 829 bp. Conserved synteny exists between the pRSB101 replication/partition (rep/par) module and the pXAC33-replicon from the phytopathogenXanthomonas axonopodispv.citri. The second pRSB101 backbone module encodes a three-Mob-protein type mobilization (mob) system with homology to that of IncQ-like plasmids. Plasmid pRSB101 is mobilizable with the help of the IncP-1αplasmid RP4 providing transfer functionsin trans. A 20 kb resistance region on pRSB101 is located within an integron-containing Tn402-like transposon. The variable region of the class 1 integron carries the genesdhfr1for a dihydrofolate reductase,aadA2for a spectinomycin/streptomycin adenylyltransferase andblaTLA-2for a so far unknown Ambler class A extended spectrumβ-lactamase. The integron-specific 3′-segment (qacEΔ1-sul1-orf5Δ) is connected to a macrolide resistance operon consisting of the genesmph(A) (macrolide 2′-phosphotransferase I),mrx(hydrophobic protein of unknown function) andmphR(A) (regulatory protein). Finally, a putative mobile element with the tetracycline resistance genestetA(tetracycline efflux pump) andtetRwas identified upstream of the Tn402-specific transposase genetniA. The second ‘genetic load’ region on pRSB101 harbours four distinct mobile genetic elements, another integron belonging to a new class and footprints of two more transposable elements. A tripartite multidrug (MDR) transporter consisting of an ATP-binding-cassette (ABC)-type ATPase and permease, and an efflux membrane fusion protein (MFP) of the RND-family is encoded between the replication/partition and the mobilization module. Homologues of the macrolide resistance genesmph(A),mrxandmphR(A) were detected on eight other erythromycin resistance-plasmids isolated from activated sludge bacteria. Plasmid pRSB101-likerepAamplicons were also obtained from plasmid-DNA preparations of the final effluents of the wastewater treatment plant indicating that pRSB101-like plasmids are released with the final effluents into the environment.

Motivation: Beyond methods for a gene-wise annotation and analysis of sequenced genomes new automated methods for functional analysis on a higher level are needed. The identification of realized metabolic pathways provides valuable information on gene expression and regulation. Detection of incomplete pathways helps to improve a constantly evolving genome annotation or discover alternative biochemical pathways. To utilize automated genome analysis on the level of metabolic pathways new methods for the dynamic representation and visualization of pathways are needed. Results: PathFinder is a tool for the dynamic visualization of metabolic pathways based on annotation data. Pathways are represented as directed acyclic graphs, graph layout algorithms accomplish the dynamic drawing and visualization of the metabolic maps. A more detailed analysis of the input data on the level of biochemical pathways helps to identify genes and detect improper parts of annotations. As an Relational Database Management System (RDBMS) based internet application PathFinder reads a list of EC-numbers or a given annotation in EMBL- or Genbank-format and dynamically generates pathway graphs. Availability: The software PathFinder will be made available on the Bielefeld Bioinformatics WebServer under the following URL: http://bibiserv.TechFak.Uni-Bielefeld.DE/pathfinder/. The source code is available upon request and will eventually be released under GPL. Contact: Alexander.Goesmann@Genetik.Uni-Bielefeld.DE

Parasite transmission strategies strongly impact host–parasite co-evolution and virulence. However, studies of vector-borne parasites such as avian malaria have neglected the potential effects of host relatedness on the exchange of parasites. To test whether extended parental care in the presence of vectors increases the probability of transmission from parents to offspring, we used high-throughput sequencing to develop microsatellites for malaria-like Leucocytozoon parasites of a wild raptor population. We show that host siblings carry genetically more similar parasites than unrelated chicks both within and across years. Moreover, chicks of mothers of the same plumage morph carried more similar parasites than nestlings whose mothers were of different morphs, consistent with matrilineal transmission of morph-specific parasite strains. Ours is the first evidence of an association between host relatedness and parasite genetic similarity, consistent with vector-mediated parent-to-offspring transmission. The conditions for such ‘quasi-vertical’ transmission may be common and could suppress the evolution of pathogen virulence.

Subseafloor sediment samples derived from a sediment core of 60m length were used to enrich psychrophilic aerobic bacteria on cellulose, xylan, chitin, and starch. A variety of species belonging to Alpha- and Gammaproteobacteria and to Flavobacteria were isolated from sediment depths between 12 and 42mbsf. Metagenomic DNA purified from the pooled enrichments was sequenced and analyzed for phylogenetic composition and presence of genes encoding carbohydrate-active enzymes. More than 200 open reading frames coding for glycoside hydrolases were identified, and more than 60 of them relevant for enzymatic degradation of lignocellulose. Four genes encoding beta-glucosidases with less than 52% identities to characterized enzymes were chosen for recombinant expression in Escherichia coli. In addition one endomannanase, two endoxylanases, and three beta-xylosidases were produced recombinantly. All genes could be actively expressed. Functional analysis revealed discrepancies and additional variability for the recombinant enzymes as compared to the sequence-based predictions.

Pseudomonas aeruginosa is a major nosocomial bacterial pathogen causing complicated catheter-associated urinary tract infections (CAUTIs). Here, we present the 6.9-Mb draft genome sequence of P. aeruginosa MH38 isolated from an acute nosocomial CAUTI. It exhibits resistance to several antibiotics but revealed low-level production of virulence factors.

The population genomics of Pseudomonas aeruginosa was analysed by genome sequencing of representative strains of the 15 most frequent clonal complexes in the P. aeruginosa population and of the five most common clones from the environment of which so far no isolate from a human infection has been detected. Gene annotation identified 5892-7187 open reading frame (ORFs; median 6381 ORFs) in the 20 6.4-7.4 Mbp large genomes. The P. aeruginosa pangenome consists of a conserved core of at least 4000 genes, a combinatorial accessory genome of a further 10 000 genes and 30 000 or more rare genes that are present in only a few strains or clonal complexes. Whole genome comparisons of single nucleotide polymorphism synteny indicated unrestricted gene flow between clonal complexes by recombination. Using standardized acute lettuce, Galleria mellonella and murine airway infection models the full spectrum of possible host responses to P. aeruginosa was observed with the 20 strains ranging from unimpaired health following infection to 100% lethality. Genome comparisons indicate that the differential genetic repertoire of clones maintains a habitat-independent gradient of virulence in the P. aeruginosa population.

Pseudomonas aeruginosa is a notable nosocomial pathogen causing severe chronic infections. Here we present the draft genome sequence of P. aeruginosa MH27, isolated from a patient with a chronic hospital-acquired catheter-associated urinary tract infection. The 7.1-Mb genome sequence organized in 24 scaffolds contributes to the understanding of biofilm formation and antibiotic resistance.

Rhizoctonia solani is a soil-borne plant pathogenic fungus of the phylum Basidiomycota. It affects a wide range of agriculturally important crops and hence is responsible for economically relevant crop losses. Transcriptome analysis of the bottom rot pathogen R. solani AG1-1B (isolate 7/3/14) by applying high-throughput sequencing and bioinformatics methods addressing Expressed Sequence Tag (EST) data interpretation provided new insights in expressed genes of this fungus. Two normalized cDNA libraries representing different cultivation conditions of the fungus were sequenced on the 454 FLX (Roche) system. Subsequent to cDNA sequence assembly and quality control, ESTs were analysed applying advanced bioinformatics methods. More than 14 000 transcript isoforms originating from approximately 10 000 predictable R. solani AG1-IB 7/3/14 genes are represented in each dataset. Comparative analyses revealed several differentially expressed genes depending on the growth conditions applied. Determinants with predicted functions in recognition processes between the fungus and the host plant were identified. Moreover, many R. solani AG1-IB ESTs were predicted to encode putative cellulose, pectin, and lignin degrading enzymes. Furthermore, genes playing a possible role in mitogen-activated protein (MAP) kinase cascades, 4-aminobutyric acid (GABA) metabolism, melanin synthesis, plant defence antagonism, phytotoxin, and mycotoxin synthesis were detected. Copyright 2014 The British Mycological Society. Published by Elsevier Ltd. All rights reserved.

Sugar beet (Beta vulgaris ssp. vulgaris) is an important crop of temperate climates which provides nearly 30% of the world's annual sugar production and is a source for bioethanol and animal feed. The species belongs to the order of Caryophylalles, is diploid with 2n = 18 chromosomes, has an estimated genome size of 714-758 megabases and shares an ancient genome triplication with other eudicot plants. Leafy beets have been cultivated since Roman times, but sugar beet is one of the most recently domesticated crops. It arose in the late eighteenth century when lines accumulating sugar in the storage root were selected from crosses made with chard and fodder beet. Here we present a reference genome sequence for sugar beet as the first non-rosid, non-asterid eudicot genome, advancing comparative genomics and phylogenetic reconstructions. The genome sequence comprises 567 megabases, of which 85% could be assigned to chromosomes. The assembly covers a large proportion of the repetitive sequence content that was estimated to be 63%. We predicted 27,421 protein-coding genes supported by transcript data and annotated them on the basis of sequence homology. Phylogenetic analyses provided evidence for the separation of Caryophyllales before the split of asterids and rosids, and revealed lineage-specific gene family expansions and losses. We sequenced spinach (Spinacia oleracea), another Caryophyllales species, and validated features that separate this clade from rosids and asterids. Intraspecific genomic variation was analysed based on the genome sequences of sea beet (Beta vulgaris ssp. maritima; progenitor of all beet crops) and four additional sugar beet accessions. We identified seven million variant positions in the reference genome, and also large regions of low variability, indicating artificial selection. The sugar beet genome sequence enables the identification of genes affecting agronomically relevant traits, supports molecular breeding and maximizes the plant's potential in energy biotechnology. This work was supported by the BMBF grant "Verbundprojekt GABI BeetSeq: Erstellung einer Referenzsequenz für das Genom der Zuckerrübe (Beta vulgaris)", FKZ 0315069A and 0315069B (to H.H. and B.W.) and by the BMBF grant/n"AnnoBeet: Annotation des Genoms der Zuckerrübe unter Berücksichtigung von/nGenfunktionen und struktureller Variabilität für Nutzung von Genomdaten in der/nPflanzenbiotechnologie.", FKZ 0315962 A, 0315962 B and 0315962 C (to B.W., H.H., and T.S.)

Motivation: Fast algorithms and well-arranged visualizations are required for the comprehensive analysis of the ever-growing size of genomic and transcriptomic next-generation sequencing data. Results: ReadXplorer is a software offering straightforward visualization and extensive analysis functions for genomic and transcriptomic DNA sequences mapped on a reference. A unique specialty of ReadXplorer is the quality classification of the read mappings. It is incorporated in all analysis functions and displayed in ReadXplorer's various synchronized data viewers for (i) the reference sequence, its base coverage as (ii) normalizable plot and (iii) histogram, (iv) read alignments and (v) read pairs. ReadXplorer's analysis capability covers RNA secondary structure prediction, single nucleotide polymorphism and deletion–insertion polymorphism detection, genomic feature and general coverage analysis. Especially for RNA-Seq data, it offers differential gene expression analysis, transcription start site and operon detection as well as RPKM value and read count calculations. Furthermore, ReadXplorer can combine or superimpose coverage of different datasets. Availability and implementation: ReadXplorer is available as open-source software at http://www.readxplorer.org along with a detailed manual. Contact: rhilker@mikrobio.med.uni-giessen.de Supplementary information: Supplementary Data are available at Bioinformatics online.

De novo genome assembly is the process of reconstructing a complete genomic sequence from countless small sequencing reads. Due to the complexity of this task, numerous genome assemblers have been developed to cope with different requirements and the different kinds of data provided by sequencers within the fast evolving field of next-generation sequencing technologies. In particular, the recently introduced generation of benchtop sequencers, like Illumina's MiSeq and Ion Torrent's Personal Genome Machine (PGM), popularized the easy, fast, and cheap sequencing of bacterial organisms to a broad range of academic and clinical institutions. With a strong pragmatic focus, here, we give a novel insight into the line of assembly evaluation surveys as we benchmark popular de novo genome assemblers based on bacterial data generated by benchtop sequencers. Therefore, single-library assemblies were generated, assembled, and compared to each other by metrics describing assembly contiguity and accuracy, and also by practice-oriented criteria as for instance computing time. In addition, we extensively analyzed the effect of the depth of coverage on the genome assemblies within reasonable ranges and the k-mer optimization problem of de Bruijn Graph assemblers. Our results show that, although both MiSeq and PGM allow for good genome assemblies, they require different approaches. They not only pair with different assembler types, but also affect assemblies differently regarding the depth of coverage where oversampling can become problematic. Assemblies vary greatly with respect to contiguity and accuracy but also by the requirement on the computing power. Consequently, no assembler can be rated best for all preconditions. Instead, the given kind of data, the demands on assembly quality, and the available computing infrastructure determines which assembler suits best. The data sets, scripts and all additional information needed to replicate our results are freely available at ftp://ftp.cebitec.uni-bielefeld.de/pub/GABenchToB.

Criblamydia sequanensis is an amoeba-resisting bacterium recently isolated from the Seine River. This Chlamydia- related bacterium harbors a genome of approximately 3 Mbp and a megaplasmid of 89,525 bp. The plasmid encodes several efflux systems and an operon for arsenite resistance. This first genome sequence within the Criblamydiaceae family enlarges our view on the evolution and the ecology of this important bacterial clade largely understudied so far.

Chinese hamster ovary (CHO) cell lines are one of the major production tools for monoclonal antibodies, recombinant proteins, and therapeutics. Although many efforts have significantly improved the availability of sequence information for CHO cells in the last years, forthcoming draft genomes still lack the information depth known from the mouse or human genomes. Many genes annotated for CHO cells and the Chinese hamster reference genome still are in silico predictions, only insufficiently verified by biological experiments. The correct annotation of transcription start sites (TSSs) is of special interest for CHO cells, as these directly define the location of the eukaryotic core promoter. Our study aims to elucidate these largely unexplored regions, trying to shed light on promoter landscapes in the Chinese hamster genome. Based on a 5' enriched dual library RNA sequencing approach 6547 TSSs were identified, of which over 90% were assigned to known genes. These TSSs were used to perform extensive promoter studies using a novel, modular bioinformatics pipeline, incorporating analyses of important regulatory elements of the eukaryotic core promoter on per-gene level and on genomic scale. Copyright 2014 Elsevier B.V. All rights reserved.

We report draft genome sequences of three Mexican Erwinia amylovora strains. A novel plasmid, pEA78, was identified. Comparative genomics revealed an rpsL chromosomal mutation conferring high-level streptomycin resistance in two strains. In the effector gene avrRpt2 , a single nucleotide polymorphism was detected that overcomes fire blight disease resistance in Malus × robusta 5.

The increasing importance of Chinese hamster ovary (CHO) cells for the production of pharmaceutical proteins has awakened the demand to understand the cellular metabolism of these cells. However, satisfactory gene expression studies have yet been impractical due to insufficient coverage of sequences. In this work, previously determined sequence information of CHO cells and newly derived data from 454 and Illumina sequencing was used to establish the CHO41K microarray which contains 41,304 probes. Self-hybridisation was performed for replica determination and samples were run in triplicates to increase statistical power. For determination of technical variance, confidence intervals at an M-value of ±0.6 for 95% and at ±2.3 for 99% of the probes were calculated. Intra-microarray and slide to slide variance was not detectable. In a first application, this microarray enabled an in-depth look inside the cellular transcriptome of CHO cells cultured in the presence or absence of the growth supporting substance "insulin like growth factor 1" (IGF-1) analogue LongR(3). Its effect on the cells ranged from enhanced growth to delay of cell death as well as cytoskeletal installation. Suggesting that under supplementation, a minimised cellular effort in installation of a large cytoskeleton occurs, possibly in favour of promoting faster cell division.

Chinese hamster ovary (CHO) cell lines represent the most commonly used mammalian expression system for the production of therapeutic proteins. In this context, detailed knowledge of the CHO cell transcriptome might help to improve biotechnological processes conducted by specific cell lines. Nevertheless, very few assembled cDNA sequences of CHO cells were publicly released until recently, which puts a severe limitation on biotechnological research. Two extended annotation systems and web-based tools, one for browsing eukaryotic genomes (GenDBE) and one for viewing eukaryotic transcriptomes (SAMS), were established as the first step towards a publicly usable CHO cell genome/transcriptome analysis platform. This is complemented by the development of a new strategy to assemble the ca. 100 million reads, sequenced from a broad range of diverse transcripts, to a high quality CHO cell transcript set. The cDNA libraries were constructed from different CHO cell lines grown under various culture conditions and sequenced using Roche/454 and Illumina sequencing technologies in addition to sequencing reads from a previous study. Two pipelines to extend and improve the CHO cell line transcripts were established. First, de novo assemblies were carried out with the Trinity and Oases assemblers, using varying k-mer sizes. The resulting contigs were screened for potential CDS using ESTScan. Redundant contigs were filtered out using cd-hit-est. The remaining CDS contigs were re-assembled with CAP3. Second, a reference-based assembly with the TopHat/Cufflinks pipeline was performed, using the recently published draft genome sequence of CHO-K1 as reference. Additionally, the de novo contigs were mapped to the reference genome using GMAP and merged with the Cufflinks assembly using the cuffmerge software. With this approach 28,874 transcripts located on 16,492 gene loci could be assembled. Combining the results of both approaches, 65,561 transcripts were identified for CHO cell lines, which could be clustered by sequence identity into 17,598 gene clusters.

Pseudomonas sp. VLB120 was isolated in Stuttgart, Germany, as a styrene degrading organism. The complete genome sequence includes genomic information of solvent tolerance mechanisms, metabolic pathways for various organic compounds, and the megaplasmid pSTY.

Background Microalgae are gaining importance as sustainable production hosts in the fields of biotechnology and bioenergy. A robust biomass accumulating strain of the genus Monoraphidium (SAG 48.87) was investigated in this work as a potential feedstock for biofuel production. The genome was sequenced, annotated, and key enzymes for triacylglycerol formation were elucidated. Results Monoraphidium neglectum was identified as an oleaginous species with favourable growth characteristics as well as a high potential for crude oil production, based on neutral lipid contents of approximately 21% (dry weight) under nitrogen starvation, composed of predominantly C18:1 and C16:0 fatty acids. Further characterization revealed growth in a relatively wide pH range and salt concentrations of up to 1.0% NaCl, in which the cells exhibited larger structures. This first full genome sequencing of a member of the Selenastraceae revealed a diploid, approximately 68 Mbp genome with a G + C content of 64.7%. The circular chloroplast genome was assembled to a 135,362 bp single contig, containing 67 protein-coding genes. The assembly of the mitochondrial genome resulted in two contigs with an approximate total size of 94 kb, the largest known mitochondrial genome within algae. 16,761 protein-coding genes were assigned to the nuclear genome. Comparison of gene sets with respect to functional categories revealed a higher gene number assigned to the category “carbohydrate metabolic process” and in “fatty acid biosynthetic process” in M. neglectum when compared to Chlamydomonas reinhardtii and Nannochloropsis gaditana, indicating a higher metabolic diversity for applications in carbohydrate conversions of biotechnological relevance. Conclusions The genome of M. neglectum, as well as the metabolic reconstruction of crucial lipid pathways, provides new insights into the diversity of the lipid metabolism in microalgae. The results of this work provide a platform to encourage the development of this strain for biotechnological applications and production concepts.

Motivation: The research area metabolomics achieved tremendous popularity and development in the last couple of years. Owing to its unique interdisciplinarity, it requires to combine knowledge from various scientific disciplines. Advances in the high-throughput technology and the consequently growing quality and quantity of data put new demands on applied analytical and computational methods. Exploration of finally generated and analyzed datasets furthermore relies on powerful tools for data mining and visualization. Results: To cover and keep up with these requirements, we have created MeltDB 2.0, a next-generation web application addressing storage, sharing, standardization, integration and analysis of metabolomics experiments. New features improve both efficiency and effectivity of the entire processing pipeline of chromatographic raw data from pre-processing to the derivation of new biological knowledge. First, the generation of high-quality metabolic datasets has been vastly simplified. Second, the new statistics tool box allows to investigate these datasets according to a wide spectrum of scientific and explorative questions. Availability: The system is publicly available at https://meltdb.cebitec.uni-bielefeld.de. A login is required but freely available. Contact: nkessler@cebitec.uni-bielefeld.de

Erwinia piriflorinigrans is a newly described pathogen causing necrosis of pear blossoms. Complete sequencing of the 37-kb plasmid pEPIR37 common to 27 E. piriflorinigrans strains revealed homology to sequences of the ubiquitous plasmids pEA29 of the fire blight pathogen E. amylovora, plasmid pEP36 of E. pyrifoliae, plasmid pEJ30 of Erwinia sp. from Japan, and genomic regions of the related Rosaceae epiphytic Erwinia species E. tasmaniensis and E. billingiae. A second 5·5-kb cryptic plasmid pEPIR5, found in 12 E. piriflorinigrans strains, was also sequenced revealing mobilization and replication proteins with similarities to many small ColE1-type plasmids in Erwinia spp. and other enterobacteria. Functional analyses of pEPIR37 introduced into a strain of E. amylovora cured of pEA29 plasmid, which has a reduced virulence, showed a role in increasing symptom development similar to that observed in E. amylovora carrying plasmid pEA29.

We reconstruct range–wide phylogeographies of two widespread and largely co-occurring Western Palearctic frogs, Rana temporaria and R. dalmatina. Based on tissue or saliva samples of over 1000 individuals, we compare a variety of genetic marker systems, including mitochondrial DNA, single-copy protein-coding nuclear genes, microsatellite loci, and single nucleotide polymorphisms (SNPs) of transcriptomes of both species. The two focal species differ radically in their phylogeographic structure, with R. temporaria being strongly variable among and within populations, and R. dalmatina homogeneous across Europe with a single strongly differentiated population in southern Italy. These differences were observed across the various markers studied, including microsatellites and SNP density, but especially in protein-coding nuclear genes where R. dalmatina had extremely low heterozygosity values across its range, including potential refugial areas. On the contrary, R. temporaria had comparably high range-wide values, including many areas of probable postglacial colonization. A phylogeny of R. temporaria based on various concatenated mtDNA genes revealed that two haplotype clades endemic to Iberia form a paraphyletic group at the base of the cladogram, and all other haplotypes form a monophyletic group, in agreement with an Iberian origin of the species. Demographic analysis suggests that R. temporaria and R. dalmatina have genealogies of roughly the same time to coalescence (TMRCA � 3.5 mya for both species), but R. temporaria might have been characterized by larger ancestral and current effective population sizes than R. dalmatina. The high genetic variation in R. temporaria can therefore be explained by its early range expansion out of Iberia, with subsequent cycles of differentiation in cryptic glacial refugial areas followed by admixture, while the range expansion of R. dalmatina into central Europe is a probably more recent event.

The plant pathogen Erwinia amylovora can be divided into two host-specific groupings; strains infecting a broad range of hosts within the Rosaceae subfamily Spiraeoideae (e.g., Malus, Pyrus, Crataegus, Sorbus) and strains infecting Rubus (raspberries and blackberries). Comparative genomic analysis of 12 strains representing distinct populations (e.g., geographic, temporal, host origin) of E. amylovora was used to describe the pan-genome of this major pathogen. The pan-genome contains 5751 coding sequences and is highly conserved relative to other phytopathogenic bacteria comprising on average 89% conserved, core genes. The chromosomes of Spiraeoideae-infecting strains were highly homogeneous, while greater genetic diversity was observed between Spiraeoideae- and Rubus-infecting strains (and among individual Rubus-infecting strains), the majority of which was attributed to variable genomic islands. Based on genomic distance scores and phylogenetic analysis, the Rubus-infecting strain ATCC BAA-2158 was genetically more closely related to the Spiraeoideae-infecting strains of E. amylovora than it was to the other Rubus-infecting strains. Analysis of the accessory genomes of Spiraeoideae- and Rubus-infecting strains has identified putative host-specific determinants including variation in the effector protein HopX1(Ea) and a putative secondary metabolite pathway only present in Rubus-infecting strains.

Streptomyces collinus Tü 365 (DSMZ 40733), isolated from Kouroussa (Guinea), is the producer of the elfamycin family antibiotic kirromycin, which inhibits bacterial protein biosynthesis by interfering with elongation factor EF-Tu. Here, we report on the Streptomyces collinus Tü 365 complete genome sequence of the 8.27 MB chromosome and the two plasmids SCO1 and SCO2.

Proteorhodopsin (PR) photoheterotrophy in the marine flavobacterium Dokdonia sp. PRO95 has previously been investigated, showing no growth stimulation in the light at intermediate carbon concentrations. Here we report the genome sequence of strain PRO95 and compare it to two other PR encoding Dokdonia genomes: that of strain 4H-3-7-5 which shows the most similar genome, and that of strain MED134 which grows better in the light under oligotrophic conditions. Our genome analysis revealed that the PRO95 genome as well as the 4H-3-7-5 genome encode a protein related to xanthorhodopsins. The genomic environment and phylogenetic distribution of this gene suggest that it may have frequently been recruited by lateral gene transfer. Expression analyses by RT-PCR and direct mRNA-sequencing showed that both rhodopsins and the complete β-carotene pathway necessary for retinal production are transcribed in PRO95. Proton translocation measurements showed enhanced proton pump activity in response to light, supporting that one or both rhodopsins are functional. Genomic information and carbon source respiration data were used to develop a defined cultivation medium for PRO95, but reproducible growth always required small amounts of yeast extract. Although PRO95 contains and expresses two rhodopsin genes, light did not stimulate its growth as determined by cell numbers in a nutrient poor seawater medium that mimics its natural environment, confirming previous experiments at intermediate carbon concentrations. Starvation or stress conditions might be needed to observe the physiological effect of light induced energy acquisition.

Hydrogen production with Chlamydomonas reinhardtii induced by sulphur starvation is a multiphase process while the cell internal metabolism is completely remodelled. The first cellular response is characterized by induction of genes with regulatory functions, followed by a total remodelling of the metabolism to provide reduction equivalents for cellular processes. We were able to characterize all major processes that provide energy and reduction equivalents during hydrogen production. Furthermore, C. reinhardtii showed a strong transcript increase for gene models responsible for stress response and detoxification of oxygen radicals. Finally, we were able to determine potential bottlenecks and target genes for manipulation to increase hydrogen production or to prolong the hydrogen production phase. The investigation of transcriptomic changes during the time course of hydrogen production in C. reinhardtii with microarrays and RNA-seq revealed new insights into the regulation and remodelling of the cell internal metabolism. Both methods showed a good correlation. The microarray platform can be used as a reliable standard tool for routine gene expression analysis. RNA-seq additionally allowed a detailed time-dependent study of gene expression and determination of new genes involved in the hydrogen production process.

Crop production may benefit from plant growth-promoting bacteria. The knowledge on bacterial communities is indispensable in agricultural systems that intend to apply beneficial bacteria to improve plant health and production of crops such as canola. In this work, the diversity of root bacterial communities associated to two different developmental phases of canola (Brassica napus L.) plants was assessed through the application of new generation sequencing technology. Total bacterial DNA was extracted from root samples from two different growth states of canola (rosette and flowering). It could be shown how bacterial communities inside the roots changed with the growing stage of the canola plants. There were differences in the abundance of the genera, family, and even the phyla identified for each sample. While in both root samples Proteobacteria was the most common phylum, at the rosette stage, the most common bacteria belonged to the family Pseudomonadaceae and the genus Pseudomonas, and in the flowering stage, the Xanthomonadaceae family and the genus Xanthomonas dominated the community. This implies in a switch in the predominant bacteria in the different developmental stages of the plant, suggesting that the plant itself interferes with the associated microbial community.

Liquid chromatography coupled to tandem mass spectrometry in combination with stable-isotope labeling is an established and widely spread method to measure gene expression on the protein level. However, it is often not considered that two opposing processes are responsible for the amount of a protein in a cell - the synthesis as well as the degradation. With this work, we provide an integrative, high-throughput method - from the experimental setup to the bioinformatics analysis - to measure synthesis and degradation rates of an organism's proteome. Applicability of the approach is demonstrated with an investigation of heat shock response, a well-understood regulatory mechanism in bacteria, on the biotechnologically relevant Corynebacterium glutamicum. Utilizing a multi-labeling approach using both heavy stable nitrogen as well as carbon isotopes cells are metabolically labeled in a pulse chase experiment to trace the labels' incorporation in newly synthesized proteins and its loss during protein degradation. Our work aims not only at the calculation of protein turnover rates but also at their statistical evaluation, including variance and hierarchical cluster analysis using the rich internet application QuPE.

The Hrp pathogenicity island (hrpPAI) of Erwinia amylovora not only encodes a type III secretion system (T3SS) and other genes required for pathogenesis on host plants, but also includes the so-called island transfer (IT) region, a region that originates from an integrative conjugative element (ICE). Comparative genomic analysis of the IT regions of two Spiraeoideae- and three Rubus-infecting strains revealed that the regions in Spiraeoideae-infecting strains were syntenic and highly conserved in length and genetic information, but that the IT regions of the Rubus-infecting strains varied in gene content and length, showing a mosaic structure. None of the ICEs in E. amylovora strains were complete, as conserved ICE genes and the left border were missing, probably due to reductive genome evolution. Comparison of the hrpPAI region of E. amylovora strains to syntenic regions from other Erwinia spp. indicates that the hrpPAI and the IT regions are the result of several insertion and deletion events that have occurred within the ICE. It also suggests that the T3SS was present in a common ancestor of the pathoadapted Erwinia spp. and that insertion and deletion events in the IT region occurred during speciation.

There was an error in the author attributions indicated in the byline. Luka Ausec and Martha Zakrzewski are co-first authors and contributed equally to this work.

DNAs of two biofilms of a thermophilic two-phase leach-bed biogas reactor fed with rye silage and winter barley straw were sequenced by 454-pyrosequencing technology to assess the biofilm-based microbial community and their genetic potential for anaerobic digestion. The studied biofilms matured on the surface of the substrates in the hydrolysis reactor (HR) and on the packing in the anaerobic filter reactor (AF). The classification of metagenome reads showed Clostridium as most prevalent bacteria in the HR, indicating a predominant role for plant material digestion. Notably, insights into the genetic potential of plant-degrading bacteria were determined as well as further bacterial groups, which may assist Clostridium in carbohydrate degradation. Methanosarcina and Methanothermobacter were determined as most prevalent methanogenic archaea. In consequence, the biofilm-based methanogenesis in this system might be driven by the hydrogenotrophic pathway but also by the aceticlastic methanogenesis depending on metabolite concentrations such as the acetic acid concentration. Moreover, bacteria, which are capable of acetate oxidation in syntrophic interaction with methanogens, were also predicted. Finally, the metagenome analysis unveiled a large number of reads with unidentified microbial origin, indicating that the anaerobic degradation process may also be conducted by up to now unknown species. 2011 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.

The complete and annotated genome sequence of Enterococcus faecalis D32, a commensal strain isolated from a Danish pig, suggests putative adaptation to the porcine host and absence of distinct virulence-associated traits.

Escherichia coli G3/10 is a component of the probiotic drug Symbioflor 2. In an in vitro assay with human intestinal epithelial cells, E. coli G3/10 is capable of suppressing adherence of enteropathogenic E. coli E2348/69. In this study, we demonstrate that a completely novel class II microcin, produced by probiotic E. coli G3/10, is responsible for this behavior. We named this antibacterial peptide microcin S (MccS). Microcin S is coded on a 50.6 kb megaplasmid of E. coli G3/10, which we have completely sequenced and annotated. The microcin S operon is about 4.7 kb in size and is comprised of four genes. Subcloning of the genes and gene fragments followed by gene expression experiments enabled us to functionally characterize all members of this operon, and to clearly identify the nucleotide sequences encoding the microcin itself (mcsS), its transport apparatus and the gene mcsI conferring self immunity against microcin S. Overexpression of cloned mcsI antagonizes MccS activity, thus protecting indicator strain E. coli E2348/69 in the in vitro adherence assay. Moreover, growth of E. coli transformed with a plasmid containing mcsS under control of an araC PBAD activator-promoter is inhibited upon mcsS induction. Our data provide further mechanistic insight into the probiotic behavior of E. coli G3/10.

This article is part of Tim Kalkhe's doctoral thesis, available in Munin at http://hdl.handle.net/10037/5248 The criteria for defining bacterial species and even the concept of bacterial species itself are under debate, and the discussion is apparently intensifying as more genome sequence data is becoming available. However, it is still unclear how the new advances in genomics should be used most efficiently to address this question. In this study we identify genes that are common to any group of genomes in our dataset, to determine whether genes specific to a particular taxon exist and to investigate their potential role in adaptation of bacteria to their specific niche. These genes were named unique core genes. Additionally, we investigate the existence and importance of unique core genes that are found in isolates of phylogenetically non-coherent groups. These groups of isolates, that share a genetic feature without sharing a closest common ancestor, are termed genophyletic groups. The bacterial family Vibrionaceae was used as the model, and we compiled and compared genome sequences of 64 different isolates. Using the software orthoMCL we determined clusters of homologous genes among the investigated genome sequences. We used multilocus sequence analysis to build a host phylogeny and mapped the numbers of unique core genes of all distinct groups of isolates onto the tree. The results show that unique core genes are more likely to be found in monophyletic groups of isolates. Genophyletic groups of isolates, in contrast, are less common especially for large groups of isolate. The subsequent annotation of unique core genes that are present in genophyletic groups indicate a high degree of horizontally transferred genes. Finally, the annotation of the unique core genes of Vibrio cholerae revealed genes involved in aerotaxis and biosynthesis of the iron-chelator vibriobactin. The presented work indicates that genes specific for any taxon inside the bacterial family Vibrionaceae exist. These unique core genes encode conserved metabolic functions that can shed light on the adaptation of a species to its ecological niche. Additionally, our study suggests that unique core genes can be used to aid classification of bacteria and contribute to a bacterial species definition on a genomic level. Furthermore, these genes may be of importance in clinical diagnostics and drug development.

Fibrella aestuarina BUZ 2 T is the type strain of the recently characterized genus Fibrella . Here we report the draft genome sequence of this strain, which consists of a single scaffold representing the chromosome (with 11 gaps) and a 161-kb circular plasmid.

Fibrisoma limi strain BUZ 3 T , a Gram-negative bacterium, was isolated from coastal mud from the North Sea (Fedderwardersiel, Germany) and characterized using a polyphasic approach in 2011. The genome consists of a chromosome of about 7.5 Mb and three plasmids.

Highlights ► We mapped all known mature CHO miRNAs to two CHO-K1 reference genomes. ► 212 unique genomic miRNA loci and the respective precursor miRNA sequences were identified. ► The genomic loci of 4 polycistronic miRNA cluster were confirmed by PCR. ► The identified sequences were analyzed for SNPs and conservation compared to mouse. ► Sequence data have been prepared for submission to miRBase miRNA sequence repository., MicroRNAs (miRNAs) have recently entered Chinese hamster ovary (CHO) cell culture technology, due to their severe impact on the regulation of cellular phenotypes. Applications of miRNAs that are envisioned range from biomarkers of favorable phenotypes to cell engineering targets. These applications, however, require a profound knowledge of miRNA sequences and their genomic organization, which exceeds the currently available information of ∼400 conserved mature CHO miRNA sequences. Based on these recently published sequences and two independent CHO-K1 genome assemblies, this publication describes the computational identification of CHO miRNA genomic loci. Using BLAST alignment, 415 previously reported CHO miRNAs were mapped to the reference genomes, and subsequently assigned to a distinct genomic miRNA locus. Sequences of the respective precursor-miRNAs were extracted from both reference genomes, folded in silico to verify correct structures and cross-compared. In the end, 212 genomic loci and pre-miRNA sequences representing 319 expressed mature miRNAs (approximately 50% of miRNAs represented matching pairs of 5′ and 3′ miRNAs) were submitted to the miRBase miRNA repository. As a proof-of-principle for the usability of the published genomic loci, four likely polycistronic miRNA cluster were chosen for PCR amplification using CHO-K1 and DHFR (-) genomic DNA. Overall, these data on the genomic context of miRNA expression in CHO will simplify the development of tools employing stable overexpression or deletion of miRNAs, allow the identification of miRNA promoters and improve detection methods such as microarrays.

The ascomycetous yeast Wickerhamomyces anomalus (formerly Pichia anomala and Hansenula anomala) exhibits antimicrobial activities and flavoring features that are responsible for its frequent association with food, beverage and feed products. However, limited information on the genetic background of this yeast and its multiple capabilities are currently available. Here, we present the draft genome sequence of the neotype strain W. anomalus DSM 6766. On the basis of pyrosequencing, a de novo assembly of this strain resulted in a draft genome sequence with a total size of 25.47 Mbp. An automatic annotation using RAPYD generated 11 512 protein-coding sequences. This annotation provided the basis to analyse metabolic capabilities, phylogenetic relationships, as well as biotechnologically important features and yielded novel candidate genes of W. anomalus DSM 6766 coding for proteins participating in antimicrobial activities.

In order to improve the genetic characterisation of the barnacle Balanus amphitrite, normalised EST libraries for the developmental stages, viz. nauplius (a mix of instars I and II), cyprid and adult, were generated. The libraries were sequenced independently using 454 technologies and 575,666 reads were generated. For adults, 4843 unique isotigs were estimated and 6754 and 7506 in the cyprid and naupliar stage, respectively. It was found that some of the previously proposed cyprid-specific bcs genes were also expressed during the naupliar and adult stage. Furthermore, as lectins have been hypothesised to influence settlement cue recognition in barnacles, the database was searched for lectin-like isotigs. Two proteins, uniquely expressed in either the cyprid or the adult stage, matched a mannose receptor, and their nucleotide sequences were 33% and 31% identical to a lectin (BRA-3) isolated from Megabalanus rosa. Further characterisation of these genes may suggest their involvement in settlement.

Motivation: The introduction of next-generation sequencing techniques and especially the high-throughput systems Solexa (Illumina Inc.) and SOLiD (ABI) made the mapping of short reads to reference sequences a standard application in modern bioinformatics. Short-read alignment is needed for reference based re-sequencing of complete genomes as well as for gene expression analysis based on transcriptome sequencing. Several approaches were developed during the last years allowing for a fast alignment of short sequences to a given template. Methods available to date use heuristic techniques to gain a speedup of the alignments, thereby missing possible alignment positions. Furthermore, most approaches return only one best hit for every query sequence, thus losing the potentially valuable information of alternative alignment positions with identical scores. Results: We developed SARUMAN (Semiglobal Alignment of short Reads Using CUDA and NeedleMAN-Wunsch), a mapping approach that returns all possible alignment positions of a read in a reference sequence under a given error threshold, together with one optimal alignment for each of these positions. Alignments are computed in parallel on graphics hardware, facilitating an considerable speedup of this normally time-consuming step. Combining our filter algorithm with CUDA-accelerated alignments, we were able to align reads to microbial genomes in time comparable or even faster than all published approaches, while still providing an exact, complete and optimal result. At the same time, SARUMAN runs on every standard Linux PC with a CUDA-compatible graphics accelerator. Availability: http://www.cebitec.uni-bielefeld.de/brf/saruman/saruman.html. Contact: jblom@cebitec.uni-bielefeld.de Supplementary information: Supplementary data are available at Bioinformatics online.

The methylotrophic yeast Pichia pastoris (Komagataella phaffii) CBS7435 is the parental strain of commonly used P. pastoris recombinant protein production hosts making it well suited for improving the understanding of associated genomic features. Here, we present a 9.35Mbp high-quality genome sequence of P. pastoris CBS7435 established by a combination of 454 and Illumina sequencing. An automatic annotation of the genome sequence yielded 5007 protein-coding genes, 124 tRNAs and 29 rRNAs. Moreover, we report the complete DNA sequence of the first mitochondrial genome of a methylotrophic yeast. Fifteen genes encoding proteins, 2 rRNA and 25 tRNA loci were identified on the 35.7kbp circular, mitochondrial DNA. Furthermore, the architecture of the putative alpha mating factor protein of P. pastoris CBS7435 turned out to be more complex than the corresponding protein of Saccharomyces cerevisiae. Copyright 2011 Elsevier B.V. All rights reserved.

Serogroup A meningococci are a leading cause of bacterial meningitis in children and young adults worldwide. However, the genetic basis of serogroup A strains' virulence and their epidemiological properties remain poorly understood. Therefore, we sequenced the complete genome of the transformable Neisseria meningitidis serogroup A strain WUE2594.

Motivation: The rapidly increasing amounts of data available from new high-throughput methods have made data processing without automated pipelines infeasible. As was pointed out in several publications, integration of data and analytic resources into workflow systems provides a solution to this problem, simplifying the task of data analysis. Various applications for defining and running workflows in the field of bioinformatics have been proposed and published, e.g. Galaxy, Mobyle, Taverna, Pegasus or Kepler. One of the main aims of such workflow systems is to enable scientists to focus on analysing their datasets instead of taking care for data management, job management or monitoring the execution of computational tasks. The currently available workflow systems achieve this goal, but fundamentally differ in their way of executing workflows. Results: We have developed the Conveyor software library, a multitiered generic workflow engine for composition, execution and monitoring of complex workflows. It features an open, extensible system architecture and concurrent program execution to exploit resources available on modern multicore CPU hardware. It offers the ability to build complex workflows with branches, loops and other control structures. Two example use cases illustrate the application of the versatile Conveyor engine to common bioinformatics problems. Availability: The Conveyor application including client and server are available at http://conveyor.cebitec.uni-bielefeld.de. Contact: conveyor@CeBiTec.Uni-Bielefeld.DE; blinke@ceBiTec.Uni-Bielefeld.De. Supplementary information: Supplementary data are available at Bioinformatics online.